The overall objective of the proposed work is to explore the application of liquid chromatography-mass spectrometry (LC-MS)-based metabolomic approaches to identify and characterize endogenous small-molecule metabolites in the peripheral system that can indicate prior chronic drug exposure or drug addiction. Because of their apparent values for revealing the status of drug abuse and monitoring the progression of drug addiction, biomarkers and biosignatures of chronic drug exposure have been pursued using biochemical, genomic, proteomic and neuroimaging approaches. However, the establishment of convincing and applicable biomarkers and biosignatures of chronic drug exposure is still a challenge. Our current proposal is based on the fact that besides the neurological changes that lead to abnormal function in the central nervous system (CNS), drug abuse and addiction also significantly affect the normal functions of the cardiovascular system, liver, kidney as well as energy metabolism, such as fatty acid and carbohydrate metabolism, through hormonal signals from CNS, transcriptional regulation or local toxicities. All these disturbances on metabolic system can definitely change the chemical composition of peripheral systems. Therefore, small-molecule biomarkers representing different aspects of chronic drug exposure can be potentially identified in the peripheral system using metabolomic approaches to examine animal models related to drug addiction. Because of its widespread abuse and harmful consequence, cocaine will be used as a prototypical compound of drug addiction in this study. In Specific Aim 1, changes in the peripheral metabolome following chronic cocaine exposure will be determined by the high-resolution LC-MS and multivariate chemometric analysis of biofluids samples collected from rats in an intermittent injection model and in a self- administration model of cocaine addiction. Biomarkers of cocaine-induced pathophysiological events will be identified by examining the metabolomic profiles of control and cocaine-treated rats in the treatment phase and withdrawal phase of both animal models. Correlation between these biomarkers and cocaine-elicited behavior will become the foundation for constructing the biosignatures of chronic cocaine exposure. In Specific Aim 2, the influence of intervention treatments on the profile of biosignatures in the cocaine-exposed rats will be examined using two mechanistically different experimental chemicals, GBR 12909 and N-acetylcysteine. In addition, the mechanism behind the cocaine-induced metabolomic changes will be probed by examining the genes, proteins and enzymes that are responsible for the generation and elimination of identified small- molecule biomarkers. PUBLIC HEALTH RELEVANCE: Metabolomic Investigation of Biosignatures of Chronic Cocaine Exposure Drug abuse can result in significant changes in the metabolic system of the body, even long after the drug use has been stopped. In this project, we will apply modern analytical technology and drug abuse animal models to identify small-molecule markers in the metabolic system that can indicate previous drug exposure. The accomplishment of this research project can contribute to the development of novel intervention and therapeutic strategies for drug abuse.